Preclinical development of complex processing systems for ex vivo culture-expanded lymphohematopoietic cells, with subsequent immunologic and/or genetic manipulation, have been carried out in collaboration with a number of National Institutes of Health investigators and with Baxter Healthcare, Inc. Preparation of CD8-depleted, culture-expanded lymphocytes, done in collaboration with Dr. Robert Walker, Dr. Michael Blaese, and Dr. Richard Morgan, involves CD8 cell depletion of peripheral blood lymphocytes using a Baxter antibody and Max Sep, an immunomagnetic system for large-scale negative selection, before ex vivo culture- expansion and gene transduction. In five large-scale runs (two preclinical and three clinical), this 10-day process resulted in a 1 to 1.5 percent depletion of CD8+ cells and 10-fold expansion of the CD4+ lymphocytes, on average. This process is currently being applied to normal syngeneic donor lymphocytes used in HIV clinical gene therapy and is potentially applicable to other clinical trials. Further process improvements and modifications are anticipated. Preparation of allogeneic donor lymphocytes selectively depleted for alloreactive T cells. This process is being developed, in collaboration with Dr. John Barrett and colleagues, for application to clinical allogeneic hematopoietic transplantation, especially in the human leukocyte antigen-mismatched setting. It involves (1) preparation of recipient (stimulator) lymphocytes, (2) preparation of donor (responder) lymphocytes, and (3) coculture and preparation of stimulator and responder lymphocytes, followed by selective depletion of alloreactive responder T lymphocytes with an anti-CD25 pseudomonas exotoxin construct. The goal is to prepare an allogeneic cellular component capable of providing normal immunologic reconstitution without concomitant graft versus host disease.